P1440: BTK INHIBITORS IMPROVE CART19 CELL THERAPY BY MODULATING IMMUNE SYSTEM
Authors: W. Luo, C. Li, M. Du, F. Zhou, Y. Zhang, L. Tang, J. Wu, H. Jiang, Q. Wei, C. Lu, H. Kou, H. Mei, Y. Hu
Journal: HemaSphere (2022)

Background: CD19-targeted chimeric antigen receptor-T (CART19) cell therapy has achieved extraordinary success in B-cell lymphoma and leukemia. However, recurrence and adverse effects, including cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), remain its obstacles. Recurrence, even resistance to CART19 cell therapy, might be attributed to CAR-T cell dysfunction and suppressor cells in the microenvironment. Cytokines required for CRS and ICANS are predominantly derived from monocytes and macrophages. Given that Bruton’s tyrosine kinase (BTK) is expressed on myeloid cells, we considered that BTK inhibitors (BTKi) could improve CAR-T cell therapy by modulating immune system. Aims: To systematically evaluate the effects of three BTKi, ibrutinib (IB), zanubrutinib (ZB) and orelabrutinib (OB), on T cells, CART19 cells and tumor microenvironment. Methods: T and CART19 cells were cultured with the three BTKi for 4 days, and the immune and differentiated phenotypes (PD1, TIM3, CTLA4, CD25, CD69 and CD28, CD45RA and CD62L) were analyzed by flow cytometry (FCM). The effects of BTKi on CART19 cytotoxicity against NALM-6 were assessed for 24-hour coculture. CART19, NALM-6 and macrophages differentiated from THP-1 upon PMA stimulation were cocultured and IL-6 in the supernatants was detected by cytometry bead assays. Lymphoma mouse models were established with BALB/c mice by subcutaneous injection of 5x106 A20 cells. IB, ZB and OB were administered to the BALB/c mice at 25, 10 and 10mg/kg respectively by oral gavage once daily for 28 days. Vehicle treatment was performed using 0.5% carboxymethylcellulose sodium. Mice in healthy controls were fed with no additional treatment. Splenocytes, bone marrow (BM) samples and tumor specimens were harvested, and single-cell suspensions were prepared to analyze immune phenotypes by FCM. Results: Compared with control T cells, IB-supplemented T cells significantly reduced the expression of PD-1 (62.28% vs 14.95%, P = 0.0024), TIM-3 (71.38% vs 7.88%, P <0.0001) and CTLA-4 (92.65% vs 15.5%, P <0.0001) following CD3/CD28 stimulation (Figure 1A). The downregulation of the suppressive markers was also observed in T cells treated with ZB, but not with OB (Figure 1A). In parallel, IB and ZB decreased activation markers expression and increased effector memory cell subsets, while decreased naïve T cells (Figure 1A and B). BTKi decreased IL-6 level in the supernatants of CART19, NALM-6 cells and macrophages (Figure 1C), and decreased CTLA-4 expression on CART19 (Figure 1D). The effects of BTKi on CART19 differentiated phenotypes were not observed (Figure E). Considering strong CD3/CD28 stimulation and tonic signaling could drive terminal differentiation and even apoptosis of CAR-T cells, we supposed BTKi could protect CART19 from excessive activation in long-term cultivation and stimulation (not shown). The three BTKi did not show synergistic effects on cytotoxicity of CART19 against NALM-6 cells (not shown). In lymphoma mice models, the three BTKi all increased the ratio of type 1 macrophages (M1) to type 2 macrophages (M2) and Th1 to Th2 in tumor, and reduced tumor-infiltrating macrophages (Figure 1F-H). BTKi induced significant decreases in exhaustion markers expression on T cells in BM tissues, but not in tumor specimens (Figure I). Image:Summary/Conclusion: Differentiation and exhaustion of CAR-T cells highly activated upon CD3/CD28 stimulation and tonic signaling can be prevented by BTKi. Moreover, BTKi can alter the tumor microenvironment and downregulate IL-6 level to improve the efficacy and safety of CART19 cell therapy.